Leo J. Grady

Identification of a Kunjin/West Nile-like flavivirus in brains of patients with New York encephalitis

Molecular analysis of brains from patients of the recent New York City encephalitis outbreak reveals the presence of a flavivirus not previously described in the Americas.

Multiple-Year Experience in the Diagnosis of Viral Central Nervous System Infections with a Panel of Polymerase Chain Reaction Assays for Detection of 11 Viruses

BACKGROUNDnPolymerase chain reaction (PCR) is becoming more common in diagnostic laboratories. In some instances, its value has been established. In other cases, assays exist, but their beneficial use has not been determined. This article summarizes findings from 3485 patients who underwent testing over a 6-year period in our laboratory.nnnMETHODSnA panel of PCR assays was used for the detection of a range of viruses associated with central nervous system (CNS) infections. PCR results were analyzed in conjunction with information about patient age and sex, the time between onset and specimen collection, and other variables. Medical chart review was conducted for 280 patients to gain diagnostic and epidemiologic insight with regard to cases of unresolved encephalitis.nnnRESULTSnA total of 498 PCR-positive samples (14.3%) were detected. Enteroviruses accounted for the largest number (360 [72.3%]) of positive PCR results, followed by herpes simplex virus (76 [15.3%]), varicella-zoster virus (29 [5.82%]), and West Nile virus (WNV) (18 [3.61%]). Of 360 patients who tested positive for enterovirus, only 46 met the Centers for Disease Control and Preventions encephalitis definition. It resulted in the greatest decrease (87.2%) in positive PCR results. Overall, the PCR positivity rate for specimens collected within 5 days after illness onset was 17.2%, compared with 8.6% for specimens collected > or =6 days after onset.nnnCONCLUSIONSnThe value of PCR in the diagnosis of viral infections has been established. PCR is of lower value in the detection of WNV in CNS, compared with serological testing, but is of greater value in the detection of other arboviruses, particularly viruses in the California serogroup. Medical chart reviews indicated that apparent CNS infection resolves in approximately 50% of cases.

Two Monoclonal Antibodies against La Crosse Virus Show Host-dependent Neutralizing Activity

Mammalian and arthropod cell cultures were used to assess the neutralizing activity of six monoclonal antibodies specific for the G1 glycoprotein of La Crosse virus. Four antibodies, two neutralizing and two non-neutralizing, showed no host-dependent differences, giving similar results when post-treatment infectivity was determined using either Aedes albopictus cells or BHK-21 cells. For two other antibodies, however, dissimilar activities were observed between the vertebrate and invertebrate cell lines. One of these antibodies was positive when BHK-21 cells were employed as the post-treatment host and negative when mosquito cells were used; the other antibody was the converse. The epitope for this last antibody was present on all California serogroup viruses examined, which suggests that it may have a special significance in the natural life-cycle of the virus.

Monoclonal Antibodies Against La Crosse Virus

Monoclonal antibodies have been used to show that an epitope is present on the G1 glycoprotein of prototype La Crosse virus that is absent or significantly altered on several isolates of La Crosse virus made in New York State, U.S.A. The portion of the G1 protein where this epitope is located plays a role in both virus neutralization and haemagglutination. Additional experiments revealed that under the appropriate assay conditions the monoclonal antibodies permitted discrimination between representatives of the North American members of the California serogroup.

The distribution of 5-bromodeoxyuridine in the DNA of polyoma-transformed mouse cells and some apparent effects on transcription☆

Abstract The DNA of polyoma-transformed mouse cells grown in the presence of 5-bromodeoxyuridine (BUdR) was examined in neutral and alkaline CsCl gradients. All of the cells had incorporated BUdR into their DNA and no distinct classes of different degrees of substitution were observed. The reassociation kinetics of DNA containing tritiated BUdR demonstrated that the analog did not preferentially replace thymidine in either the repetitive or non-repetitive sequences. RNA-DNA saturation hybridization experiments using RNA extracted from cells grown in medium containing BUdR revealed an apparent reduction in the extent of non-repetitive DNA transcription compared with untreated control cells. The degree to which regulatory genes, as opposed to structural genes, is involved in this decrease cannot be determined from these data.

Evidence that fatal human infections with La Crosse virus may be associated with a narrow range of genotypes

La Crosse (LAC) virus belongs to the California (CAL) serogroup of the genus Bunyavirus, family Bunyaviridae. It is considered one of the most important mosquito-borne pathogens in North America, especially in the upper Mid-West, where it is associated with encephalitis during the time of year when mosquitoes are active. Infections occur most frequently in children and young adults and, while most cases are resolved after a period of intense illness, a small fraction (< 1%) are fatal. At present there have only been three isolates of LAC virus from humans all made from brain tissue postmortem. The cases yielding viruses are separated chronologically by 33 years and geographically from Minnesota/Wisconsin (1960, 1978) to Missouri (1993). The M RNA sequence of the first two isolates was previously reported. The present study extends the observations to the isolate from the 1993 case and includes several mosquito isolates as well. A comparison of the M RNAs of these viruses shows that for the human isolates both nucleotide sequence and the deduced amino-acid sequence of the encoded proteins are highly conserved, showing a maximum variation of only 0.91% and 0.69%, respectively. This high degree of conservation over time and space leads to the hypothesis that human infections with this particular genotype of LAC virus are those most likely to have a fatal outcome. It is also shown that a virus with this genotype could be found circulating in mosquitoes in an area more or less intermediate between the locations of the first and second fatal cases.

Diagnosis of Jamestown Canyon Encephalitis by Polymerase Chain Reaction

In recent years, polymerase chain reaction (PCR) has been under study as a potential technique to improve the accuracy of diagnosis of suspected central nervous system viral infections. We describe a case of severe encephalitis in a previously healthy 20-year-old woman from New York who presented with headache, fever, and photophobia. Her illness was characterized by progressive worsening of her neurological status, leading to confusion, delirium, and status epilepticus. The diagnosis of Jamestown Canyon encephalitis was established by positive reverse transcriptase (RT)-PCR and nucleic acid sequencing of the band from both cerebrospinal fluid and brain tissue. The nucleotide sequence and the deduced amino acid sequence of the Jamestown Canyon virus from this patient were very similar to Jamestown Canyon virus isolates from mosquito pools in New York. This report suggests that RT-PCR assays could be important tools in the diagnostic workup of cases of encephalitis.

Evidence for three separate antigenic sites on the G1 protein of La crosse virus

Competitive binding assays with monoclonal antibodies have been used to show that there are a minimum of three nonoverlapping antigenic sites on the G1 glycoprotein of La Crosse virus. One of these sites contains the epitopes for three of the five monoclonal antibodies employed and is involved with both hemagglutination and neutralization. A second site contains the epitope for an antibody that inhibits hemagglutination but has no neutralizing activity. The third site encompasses the epitope for an antibody that at present has no identified biological role.

A comparison of several procedures for reducing RNase contamination in preparations of DNase and a particularly successful combination of methods.

Abstract The three methods most commonly used for removing RNase from preparations of DNase were compared under similar assay conditions for residual RNase activity. In order of their effectiveness they ranked: (1) bentonite, (2) agarose-UMP, (3) iodoacetate. Even the best of the single treatments did not eliminate all of the RNase, and it was discovered that a significant improvement was obtained when DNase was first passed over agarose-UMP and then treated with bentonite.

Molecular complexity of vaccinia DNA and the presence of reiterated sequences in the genome

Abstract Reassociation rate measurements suggest that the molecular weight of the DNA of vaccinia virus is 1.2 × 10 8 , when polyoma virus DNA is used as a standard of comparison. In addition, a search for reiterated sequences revealed that a small portion of the genome (0.6%) occurs as multiple copies.